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Journal of Drug Delivery and Therapeutics

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Identification of potential inhibitors of SARS-CoV-2 from Artemisia annua compounds by In silico evaluation and their density functional theory (DFT)

Abdirahman ELMI¹*, Ahmed Said MOHAMED¹, Nazia SIDDIQUI², Syad AL JAWAD³, Moustapha NOUR⁴, Idriss MIGANEH¹ and Saleem JAVED⁵*

¹ Medicinal Research Institute, Centre d’Etudes et de Recherche de Djibouti, IRM-CERD, Route de l’Aéroport, Djibouti

²USIC, Dayalbagh Educational Institute, Agra, India 282005

³International Islamic University Chittagong, Department of pharmacy, Bangladesh

⁴ Centre d’Etudes et de Recherche de Djibouti, ISV-CERD, Route de l’Aéroport, Djibouti

⁵Department of Chemistry, Institute of H. Science, Dr. B. R. Ambedkar University, Agra, India 282002

1     INTRODUCTION

Artemisia is one of the most widespread genera in the Asteraceae family. It is a heterogeneous genus, composed of more than 500 different species distributed mainly in the temperate zones ¹.

A large number of studies have been carried out to determine precisely the chemical compositions of the Artemisia species, especially for the two species A. annua and A. afra which have high medicinal value. Sesquiterpene lactones, phenolic compounds and flavones have been identified in A. annua ^2–7 and A. afra contains monoterpenoids, sesquiterpenes, glaucolides, guaianolides, flavonoids ⁸.

Also another study showed that A. annua has an antiviral potential greater than that of 20 other medicinal plants tested. The good antiviral activity of A. annua can be explained by the presence of sterols having better antiviral activity than artemisinin or artanuin-B, two caracteristic molecules for this species ¹⁰.

Quercetin is also one of the naturally occurring substances found in Artemisia A. annua shown to be effective for a wide range of antiviral use ¹¹.

Recently, in April 2020, the Madagascan president promoted the name of an anti-Covid remedy, called "The Covid-Organics" (CVO): an improved traditional remedy based on Artemisia and medicinal plants from Madagascar. Combined with conventional therapies, herbal teas show an attenuation of respiratory symptoms. It is possible that molecules of A. annua play a role in blocking the two main receptors (serum protease, ACE2 receptor) of the cell membrane, i.e. the two main keys that allow the virus to access the cell  (Artemisia and Covid-19: the remedy Malagasy boosts Africa, Paris Match, Published on 05/18/2020).

It is known also that the A. annua has always been regarded as a reference to clear heat and eliminate dampness. For this reason, A annua acts as a pivotal medicine in the prescription of treatment of SARS ¹³. Similar, to SARS, Covid-19 is also a coronavirus that causes respiratory syndromes, which startith dampness, with a long latency and courses. The pathogenesis of Covid-19 is mainly characterized by the inward invasion of dampness towards the transformation into heat. Covid-19 treatment should focus on clearing heat, removing dampness and resolving phlegm. Considering the initial clinical manifestations of Covid-19 characterized by pathogen invading Shaoyang gallbladder meridian, A annua is expected to be an essential drug for the treatment of Covid-19 ¹⁴.

The natural product tested against this coronavirus reacted largely well against the spike glycoprotein (S) on the surface of SARS-CoV-2 precisely, by binding the domain of SARS-CoV-2 spike protein and SARS-CoV-2 main protease ^{14, 15}. In this present study we also targeted furin, a kind of proprotein convertases. Nine compounds from the Artemisia annua are tested by Insilico for their affinity with these three targets. Molecular simulation by density functional theory is carried out on the compounds with the best bond energies.

2     MATERIALS AND METHODS

2.1 Compounds tested

The genus Artemisia is rich in phytocompounds and the presence of three major families of secondary metabolites has been reported ¹⁶. There are basically nine compounds found in the Artemisia annua. They have been stimulated to determine their anti SARS-Cov-2 potential. These compounds are: Artemisinin, Arteannuin B, Alpha Thujone, P-hydroxyacetophenone, Fisetin, Cirsimaritin, Capillin, β-Sitosterol and Quercetin.

2.2 Docking targets

The targets are SARS-CoV-2 main protease (Mp), SARS-CoV-2 receptor binding domain (RBD) and human furin protease (HF protease). These targets have  a crucial role in the virus propagation ^{14, 15}. In addition SARS-CoV-2 Mp is present in most viruses of the coronavirus family and therefore its functioning is widely described in the literature ¹⁷.

2.3 Docking protocol

The description of molecular docking, prediction of pharmacokinetic parameters as well as the toxicological property was carried out in our previous article ¹⁸.

2.4 Density Functional Theory

Density Functional Theory (DFT) is currently the most successful and promising quantum chemical approach to calculate ground state properties of atoms and molecules. In this paper, all calculations were done by using the B3LYP method and 6-311++G(d,p) basis set ^{18, 19}. The computations for the present work has been performed using the Gaussian 03W ²⁰ and ORCA 4.0.1 ²¹. All vibrational wavenumbers and geometrical parameters were computed as decrypted in ²². The topological parameters were determined using Multiwfn software ²³. All the graphs were plotted using Origin 8.0 software²⁴ or Multiwfn software.

3     RESULTS AND DISCUSSIONS

3.1 Bioavailability parameters and Toxicity of selected compounds

When designing a drug, the study of the physico-chemical parameters of the candidate molecule is essential. These parameters predict the behavior of the compound during its oral administration and its bioavailability ²⁵. Two rules govern the analysis of these parameters: Lipinski and Veber. According to the first rule, the candidate molecules must have a value equal or multiple of 5. The optimal properties and the corresponding values are: number of active hydrogen atoms (HBD> 5), number of binding sites for hydrogen atoms (HBA> 10), logarithm of the octanol / water partition (LogP< 5), molecular weight (MW <500 DA) ²⁶. Beyond two unsatisfied conditions, a candidate molecule is discarded in the drug research process. Our nine compounds from Artemisia annua fulfill these two rules (Table A, Support information).

Also, a predictive evaluation of the possible toxicity of these compounds was made with four different parameters namely Ames toxicity, Carcinogens, Acute oral toxicity and Rat acute toxicity. They have low toxicity whatever the parameters considered (Table B, Support information).

3.2 Molecular docking

The search for therapeutic molecules is carried out in different stages. Molecular modelling makes it possible to pre-select the candidate molecules for a given target. The hydrogen bond between the ligand (therapeutic molecule) and the protein/enzyme of the pathogen is studied and a binding energy is measured. A low binding energy corresponds to a good affinity between the ligand and the biological target.

Binding energies range from -2.519 for Artemisinin at the HF protease site to -7.169 for Quercetin at the SARS-CoV-2 Mp site (Table 1). On the SARS-CoV-2 RBD target site, six compounds present in Artesimia sp (66% of compounds tested) have better binding energies than hydroxychloroquine which is very widely used against Covid-19. And this SARS-CoV-2 RBD target is interesting since this part allows this virus to attach to the host cell. By blocking this receptor with inhibitors like these biomolecules, the virus will not be able to attach itself to cells. Recalling, moreover, that the virus is essentially made up of proteins surrounding genetic material and needs the cellular tools to multiply ²⁷.

Compounds with a better binding energy than one of the reference drugs are shown in 2D to better visualize their interactions with the amino acid residues of the targets (Figure 1). Quercetin, having a better score than the two references, is represented in 3D (Figure 2).

The first two targets (SARS-CoV-2 Mp and SARS-CoV-2 RBD) located on the virus Artemisinin have a better binding energy, twice as small as that of the third human furin protease target, -4.825, -4.916 and -2.519 kcal/mol respectively. The action of this active compound in A annua would be directed towards the virus rather than host cell protection. This observation is applicable to its derivative Arteannuin B which has binding energies -5.075 and -5.604 and -3.342 kcal/mol at the SARS-CoV-2 Mp, SARS-CoV-2 RBD and human furin protease sites respectively. Against SARS-CoV-2 RBD these two compounds are better than hydroxychloroquine (Table 1).

However, on the three target sites, Quercetin and Fisetin have the best affinities (Table 1). The antiviral action of Quercetin is already obtained against HIV ²⁸, against influenza virus ²⁹, herpes virus ³⁰ and against the dengue virus ³¹. A recent study has shown that this compound can interfere with 85% of the functions of the SARS-Cov-2 viral proteins inside human cells. This value rises to 93% if Quercetin is combined with vitamin D ³².

Figure 1: 2D visualization of molecular interaction of SARS-CoV-2 Mp (A), SARS-CoV-2 RBD (B) and human furin protease (C) with the biomolecules having at least better binding energy than one of standard.

Table 1: Molecular docking of biomolecules with SARS-CoV-2 Mp, SARS-CoV-2 RBD and human furin protease.

*Remdesivir and Hydroxychloroquine used as references.

The two flavonols which have the best scores in docking, Quercetin and Fisetin, are classified in category II for Acute oral toxicity with an LD50 between 50 mg/kg and 500 mg/kg (Table 1). Although the two compounds have very similar structures, the OH group at the 5th position on the A ring and the B ring as well as the hydroxyl number differ. Quercetin has five hydroxyl groups and 4 for Fisetin. Also, the latter has an OH at 5 ’of the B ring. The structure-activity study of different flavonoids has shown that the presence of an OH at the 5'B ring decreases the inhibitory action of polyphenols ³³. On the other hand, OH in the A ring, as is the case with Quercetin, is beneficial in the action against rhinovirus ³⁴.

Figure 2: 3D visualization of docking analysis of human furin protease binding with quercetin, better binding energy than the two standards.

On the first two targets, the amino acids that the two flavonols interact with are similar. ASN142; HIE163 bond with Fisetin and for Quercetin ASN142; HIE163; GLU166; HIE164; GLN189 on SARS-CoV-2 Mp (Table 1). Likewise, Fisetin bond with LEU390 and ASN388 on SARS-CoV-2 RBD and Quercetin bond with ILE358 and ASN388. On the other hand, against human furin protease, the binding amino acids are totally different for the two flavonols: ALA292, PRO256, ASP258 for fisetin and LEU227, GLU236, ASH264 for quercetin. In the latter target, the two compounds can be complementary. On this same target, quercetin is better than the reference drugs, remdesivir and hydroxychloroquine (Table 1).

The structural study in particular Density Functional Theory of these two flavonols is crucial to have the possible link of their action with the SARS-CoV-2 virus.

3.3 Density Functional Theory of Quercetin and Fisetin

3.3.1            Optimized molecular geometry for quercetin and fisetin

TheB3LYP /6-311++G(d,p) optimized structure of both the molecules Fisetin and Quercetin along with their atom numbering  are presented in figure 3. Optimized bond lengths and angles are mentioned in Table C and D (Support Information). The studied compounds retain a C1 point group. The optimized geometry of the studied compounds is in the range of expected bond lengths and bond angles. The C-C, C-O, C-H and O-H bond lengths are in the regular order. C-C-C bond angles in the ring and C-C-O along with C-O-H showed well expected bond angles as given in Table C and D (Support Information).

Fisetin                                                                                                   Quercetin

Figure 3: Optimized geometric structure with atom numbering of Fisetin (left) and Quercetin (right), brown-coloured are carbon and red are nitrogen.

Bond lengths of C=O (R 28-31) in Fisetin is 1.2393 Å while in Quercetin (R 16-17) is 1.2426 Å. The O-H bond lengths (R 10-11, 29-30, 26-27 and 24-25) in Fisetin range from 0.9626 to 0. 9798 Å and the O-H bond lengths (R 9-10, 31-32, 27-28 and 29-30) in Quercetin range from 0.9626 to 0.9932 Å.

3.3.2            Vibrational spectral analysis

The vibrations frequencies, Raman and IR spectrum analysis of both Fisetin and Quercetin compounds have been obtained by using B3LYP/6-311++G(d,p) basis set along with small scaling factor 0.961. The fact that no imaginary frequencies were found in prediction implies that the optimized geometry is located at the local lowest point on the potential energy surface.

Table 2: The values of calculated dipole moment μ(D), polarizability (α₀), first order hyperpolarisability, (β_tot) components of Fisetin and Quercetin.

Fisetin has 31 atoms and 87 fundamental vibrations while Quercetin has 32 atoms and 90 fundamental vibrations, both showed C_S and C₁ point group symmetry. The calculated IR and Raman frequencies together with relative intensities are presented in Table E and F (Support Information). C=O in Fisetin and Quercetin appears at 1612 and 1622 cm^-1 respectively. All C-H and C-C bonds vibrations are in the expected range in both the molecules.

The IR spectrum of quercetin shows an intense and thin band at approximately 3300 cm^-1, characteristic of bound –OH groups. However, the signatures of -OH bound in Fisetin appear at approximately 3500 cm^-1. These differences in intensity and vibration are due to the diversity in the number of –OH groups and the chemical environment of the two molecules respectively. These bands can be explained by the formation of intramolecular hydrogen bonds, generally of the type C = O--- HO– and –OH---OH. The number of hydroxyl groups as well as their positions can be an influencing factor for the anti-Covid activity against intermolecular interactions (hydrogen-bridge) between Fisetin or Quercetin and virus. Calculated IR and Raman Spectra are displayed in Figure. 4 and 5 for Fisetin and Quercetin.

Figure 4: Infrared spectra of Fisetin(left) and Quercetin (right) using DFT/6-311++G(d,p)

         

Figure 5: Calculated computational Raman spectra of Fisetin(left) and Quercetin (right) using DFT/6-311++G(d,p)

3.3.3            Molecular electrical potential surface

The contribution of charges may be presented in 3D by utilizing molecular electrical potential (MEP). This prediction facilitates the recognition of interactions of molecule and chemical bond nature. It displays the molecular size, shape, electrostatic potential charge through colour grading. Thus, various physicochemical properties of a molecule can be determined by MEP ³⁵, like, interaction of nucleic acids with their constituent bases; enzyme-ligand or protein-ligand interactions among others ³⁶. The different colour in Figure 6 shows distinct values of the electrostatic potential at the surface of the compounds.

The electrostatic potential is arranged from smallest (red) to largest (blue). Here, we are showing deepest red for the negative charge and deepest blue for the positive charge. The colour code of the maps is found to be in the range of (deepest red) -1.70 to +1.70 eV (deepest blue), for the Fisetin and -1.381(deepest red) to + 1.381 eV (deepest blue) for the Quercetin molecule. The red colour corresponds electrophilic attack and the blue colour shows the nucleophilic attack.

Figure 6: Molecular electrostatic potential (MEP) of Fisetin(left) and Quercetin (right)

3.3.4            Non-Linear Optical (NLO) property analysis

The NLO properties of Fisetin and Quercetin were investigated by the DFT/B3LYP technique to establish the NLO character from polarizability, hyperpolarisability and dipole moment calculations. The value of hyperpolarisability have a strong sensitivity to the electron^36–38.

The dipole moment shows highest value at 1.84 and 1.734 D for Fisetin and Quercetin respectively. The values are tabulated in Table 2. The value of the dipole moment increases with the intramolecular interactions.

The hyperpolarisability value for the Fisetin and Quercetin are 9.26 x 10^-31 and 1.89 x 10^-30 esu and this large value of the two molecules implies that they have significant NLO properties.

3.3.5            UV-Vis spectral analysis and Frontier molecular orbital

The UV-Vis spectrum of the Fisetin and Quercetin as represented in Figure 7 was computed in gas phase. The electronic transition values for both the molecules are gathered in Table 3. In gas phase, the absorption wavelengths of Fisetin and Quercetin are detected at 352, 303 and 356, 307 nm. These values are similar in gas and solvent phase. Therefore the solvent has no effect on the optical activity of the molecule.